Astaxanthin is known to be distributed in a wide variety of organisms, such as animals (e.g. birds such as flamingo and scarlet ibis, and fish such as rainbow trout and salmon), algae and microorganisms. It is also recognized that astaxanthin has a strong antioxidation property against active oxygen as well as most carotenoids. This property is expected to apply to the pharmaceutical usage of astaxanthin to protect living cells against some diseases, such as a cancer. Moreover, from the industrial application viewpoint, a demand for astaxanthin as a coloring reagent is increasing, especially in the farmed fish industry, such as salmon, because astaxanthin imparts distinctive orange-red coloration to the animals and contributes to consumer appeal in the marketplace.
Phaffia rhodozyma is known as a carotenogenic yeast strain that produces astaxanthin specifically. Different from the other carotenogenic yeast, Rhodotorula, Phaffia rhodozyma can ferment some sugars such as D-glucose. This is an important feature from an industrial application viewpoint. In a recent taxonomic study, a sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated under the name of Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast 11, 101-110, 1995). Some strain improvement studies to obtain hyper-producers of astaxanthin from P. rhodozyma have been conducted, however, in this decade, such effort have been restricted to employing the method of conventional mutagenesis and protoplast fusion. Recently, Wery et al. developed a host vector system using P. rhodozyma in which a non-replicable plasmid was used for multicopy integration onto the genome of P. rhodozyma at the ribosomal DNA locus (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported the use of improved vectors to obtain a transformant of P. rhodozyma, as well as its three carotenogenic genes which code the enzymes that catalyze the reactions from geranylgeranyl pyrophosphate to beta-carotene (International patent WO97/23633). The importance of genetic engineering methods to P. rhodozyma strain improvement studies should increase in the near future in order to surpass the productivity levels reached by conventional methods.
As described above, astaxanthin has an antioxidant property. This feature seems to have an important role in vivo for a protecting against active oxygen species such as O2.,H2O2 and OH., which are continuously generated in living cells. An et al. obtained a hyperproducer of astaxanthin from P. rhodozyma by selecting for an antimycin-sensitive strain after conventional chemical mutagenesis (An, G-H. et al., Appl. Env. Microbiol., 55 (1), 116-124, 1989). Antimycin is known to be an inhibitor of respiratory chain between cytochrome b and C1 (Lucchini, G. et al., Mol. Gen. Genet., 177, 139-, 1979) and such antimycin-sensitive mutants display enhanced pigmentation. Furthermore, active oxygen species produced due to a blockade of the primary respiratory chain at the bc1 complex stimulated carotenoid formation (An, G-H et al., Appl. Env. Microbiol., 55, 116-124, 1989). Indeed, addition of an O2. generator, duroquinone, to the growth medium increased total carotenoid content (the main carotenoid is astaxanthin) as well as the relative amounts of xanthophylls present in P. rhodozyma, while the active oxygen species-quenching factor mannitol reversed this effect (Schroeder, W. A. et al., J. Gen. Microbiol., 139, 907-912, 1993). These results prompted the authors to speculate on the antioxidant property of astaxanthin in P. rhodozyma. In fact, astaxanthin production is stimulated in post-exponential growth phase when respiration activity is fully induced. Moreover, the addition of respiratory substrate, such as ethanol, to the medium enhanced astaxanthin production in P. rhodozyma (Gu, W-L. et al., J. Ind. Microbiol. Biotechnol., 19, 114-117, 1997). Schroeder et al. tried to determine the relationship of the superoxide dismutase (SOD) and catalase activities, which act as native active oxygen species-quenching factors in P. rhodozyma to the productivity of astaxanthin, by comparing the difference between a parent strain and an antimycin-sensitive hyperproducer of astaxanthin. However, direct correlation of in vitro activity could not be observed.